Performance of six diagnostic tests to screen for Chagas disease in blood banks and prevalence of Trypanosoma cruzi infection among donors with inconclusive serology screening based on the analysis of epidemiological variables

ORIGINAL ARTICLE

Performance of six diagnostic tests to screen for Chagas disease in blood banks and prevalence of Trypanosoma cruzi infection among donors with inconclusive serology screening based on the analysis of epidemiological variables

Gilberto de Araujo Pereira^I; Francisco Louzada-Neto^II; Valdirene de Fátima Barbosa^I; Márcia Maria Ferreira-Silva^I; Helio de Moraes-Souza^I

^IUniversidade Federal do Triângulo Mineiro - UFTM, Uberaba, MG, Brazil

^IIUniversidade de São Paulo - USP, São Carlos, SP, Brazil

^{Corresponding author}

ABSTRACT

OBJECTIVE: The frequent occurrence of inconclusive serology in blood banks and the absence of a gold standard test for Chagas'disease led us to examine the efficacy of the blood culture test and five commercial tests (ELISA, IIF, HAI, c-ELISA, rec-ELISA) used in screening blood donors for Chagas disease, as well as to investigate the prevalence of Trypanosoma cruzi infection among donors with inconclusive serology screening in respect to some epidemiological variables.

METHODS: To obtain estimates of interest we considered a Bayesian latent class model with inclusion of covariates from the logit link.

RESULTS: A better performance was observed with some categories of epidemiological variables. In addition, all pairs of tests (excluding the blood culture test) presented as good alternatives for both screening (sensitivity > 99.96% in parallel testing) and for confirmation (specificity > 99.93% in serial testing) of Chagas disease. The prevalence of 13.30% observed in the stratum of donors with inconclusive serology, means that probably most of these are non-reactive serology. In addition, depending on the level of specific epidemiological variables, the absence of infection can be predicted with a probability of 100% in this group from the pairs of tests using parallel testing.

CONCLUSION: The epidemiological variables can lead to improved test results and thus assist in the clarification of inconclusive serology screening results. Moreover, all combinations of pairs using the five commercial tests are good alternatives to confirm results.

Keywords: Blood donors; Chagas disease; Sensitivity and specificity; Epidemiologic factors

Introduction

Chagas disease, originally described by the Brazilian researcher, Carlos Justiniano Ribeiro Chagas, in 1909 is one of the most widely distributed infectious diseases on the American continent, especially in Latin America, with approximately 25 million people living in risk areas. An estimated 10 million individuals are infected worldwide mostly in Latin America⁽¹⁾. The gradual control of natural transmission, mainly due to eradication of the vector in various countries where the disease is endemic, demonstrates the presence of secondary mechanisms of Chagas disease transmission, especially transfusional transmission^(2-5).

Scientific-technical progress in the fight against Chagas disease intensified in the 1980s. Thus, the intensification and standardization of sanitary surveillance measures by public andprivate blood centers in endemic countries - that began in the late 1960s in some Latin Americancountries - markedly contributed to a reduction in the frequency of non-negative serology forTrypanosoma cruzi among blood donors. While in the 1980s the predominance of seropositivedonors in Latin America was 6%, this index dropped to 1.28 % by 2006⁽²⁾. Concomitantly,transfusional transmission cases of Chagas disease have occurred in North America, Europe,Japan and Australia^(1,4). Thus Chagas disease, which until recently had been seen as a seriousLatin-American public health problem, has become a global public health threat. Especially withrespect to the quality control of serological screening for Chagas disease among blood donors,the occurrence of false-positive results may lead to unnecessary disposal of blood units andconsequently compromise the blood supply at blood centers. In addition, there are psychologicaland social consequences for the ineligible donor who believes that he has a stigmatized chronicdisease. On the other hand, the occurrence of false-negative results may lead to transfusionaltransmission of Chagas disease. The greatest challenge currently encountered by blood banksis the frequent occurrence of inconclusive reactions, most of which are observed among nonchagasic donors; this indicates failure in the specificity of screening tests^(6-8).

In view of the lack of consensus in the international literature regarding the diagnostic test that correctly classifies a subject as seropositive or seronegative for Chagas disease (100% sensitivity and specificity), numerous researchers have proposed to improve the existing variations of the enzyme-linked immunosorbent assay (ELISA) - one technique recommended by ANVISA, the Brazilian National Heath Surveillance Agency for screening donors - for example, by replacing natural T. cruzi antigens with recombinant antigen mixtures^(9-12).

Several investigators have also proposed the inclusion of aclinical-epidemiological chart to assist in the investigation anddefinition of the true serological profile of ineligible donors,especially those presenting inconclusive reactions^(7,13-15). This chart would include questions regarding the place and type of residence, afamily history of Chagas disease, a history of contact with the vector,and a history of blood transfusions and surgery. This procedure hasbeen shown to be effective in the differentiation between donors with or without T. cruzi infection when used simultaneously withserological and/or molecular biology tests^(7,14), and will certainly bean important tool for the selection and exclusion of blood donorsat blood centers in countries or geographic regions where Chagasdisease is not endemic⁽¹⁵⁾.

Thus, the goal of this work is to evaluate the efficacy of the blood culture test and five commercial tests (ELISA, IIF, HAI, c-ELISA, rec-ELISA) used in screening blood donors for Chagas disease, as well as to investigate the prevalence of T. cruzi infection among donors with inconclusive serology screening in respect to some epidemiological variables.

Methods

The target population of this study was stratified from a total of 95,990 donations based on a retrospective study ofthe database of the Uberaba Regional Blood Center (URBC)conducted between January 2000 and December 2005. From 269non-negative donors for Chagas disease (0.28%), 60 participantswere randomly selected: Stratum II: 30 donors repeatedly positiveusing two screening tests (ELISA and IIF) and Stratum III: 30donors with inconclusive serology in the ELISA screening testor with discordant results in two tests. Additionally 30 donorswith more than five negative donations at URBC over a six-yearperiod (Stratum I) were included. This number was fixed due to thedifficulty in contacting ineligible donors, especially those ineligiblebefore 2003, which led to sample loss. The main reasons for this losswere changes of address and telephone number (30.4%), residingmore than 120 km from Uberaba (26.4%), refusal to participatein the study (7%) and death (2%). All participants answered thefollowing socio-epidemiological questionnaire: type of donor (firsttime, repeat), age (< 30: > 30 years), gender (male: female), livingin an endemic region (yes: no), contact with the vector transmittingChagas disease (yes: no) and a family history of Chagas disease(yes: no). A 30-mL sample of blood was drawn for each participantto perform five serological tests and a parasitological test.

In addition to the commercial ELISA test (ELISA cruzi^® bioMérieux Diagnostica SA, Rio de Janeiro, Brazil), five other immunodiagnostic screening tests were performed in theninety donors invited to participate in the study including: thecommercial ELISA test (ELISA Chagatest^® Wiener Lab, Rosario, Argentina); indirect immunofluorescence test (IIF - Imunocruzi^®, bioMerieux Diagnostics SA, Rio de Janeiro, Brazil); indirecthemagglutination (IHA - Chagatest^®, Wiener Lab, Rosario,Argentina); conventional ELISA (c-ELISA) which uses antigensderived from T. cruzi lysate and recombinant ELISA (rec-ELISA) using the recombinant antigens cytoplasmic repetitive antigen (CRA) and flagellar repetitive antigen (FRA) (EIE-Recombinant-Chagas Bio-Manguinhos^®, Bio-Manguinhos Laboratory, Oswaldo Cruz Foundation / FIOCRUZ, Brazilian Ministry of Health) and the blood culture test (HEMO), the only test that is known to be 100% specific as it demonstrates the presence of the parasite.

Statistical Analysis

Due to the absence of a gold standard for the diagnosis ofChagas disease, a Bayesian latent class statistical model was considered in this particular case of six diagnostic tests, six covariatesin the logit model^(16,17) considering the assumption proposed by Hui& Walter⁽¹⁸⁾, with different prevalence rates for chagasic infectionbut with a similar test performance among strata (SI: negative,SII: positive and SIII: inconclusive serology in the screening). Thenumerical Bayesian algorithm (Metropolis-Hastings algorithm) andthe convergence evaluation were implemented in package R, whichis freely available from www.r-project.org. The codes designed forthe present study can be requested by e-mail from the authors.

Ethical Aspects

The study was approved by the Ethics Committee of Universidade Federal do Triângulo Mineiro (UFTM - protocol # 464) and all participants signed consent forms.

Results

Table 1 shows the results of the six tests under investigation for each of the three strata according to the result of serologicalscreening at the time of donation. It was observed that 90% of donors with negative and 80% with inconclusive screening results were negative in all six tests investigated. Among the 30 donors with positive screening results, 96.6% had five or six positive results in the tests under investigation.

On analyzing the performance of tests without considering the covariates and the blood culture test, the general sensitivity between tests is very similar and higher than 97.63%. Two tests (IHA and IIF) were more specific with rates of 98.48% and 98.52%, respectively (Table 2).

There was an increase in these rates when the covariates were taken into account, for example, all tests had sensitivities equal to orgreater than 99.05% for donors older than 30 years and with a historyof Chagas disease in the family; the ELISA, IIF and IHA tests hadspecificity rates above 99.05% for these same categories (Table 2).

Despite the very similar nominal values, the sensitivity rates of all serological tests (ELISA, IIF, IHA, c-ELISA and rec-ELISA) were higher in over 30-year-old donors, females, who came from an endemic region, who had had contact with the vector and had a family history of Chagas disease. The IIF, IHA, c-ELISA and rec-ELISA tests were found to be more specific in repeat donors, over 30-year-old donors, females, who came from an endemic region, who had not had contact with the vector and had a family history of Chagas disease. In contrast, the ELISA test was more specific in first time donors who had had contact with the vector transmitting the disease (Table 2).

With respect to the prevalence of Chagas disease, an overallestimate of 13.30% was observed among donors with inconclusiveserology in the screening test at the time of blood donation but thisrate varied considerably depending on the covariates (Table 3).

When we consider testing in series which is suitable for situations that require greater specificity, where the diagnostic result is positive if all tests have positive results, we find a specificity of over 99.93% for all pairs of tests (not including the blood culture test) and 100% when we consider the ELISA, IIF, IHA tests together. While, the parallel scheme, indicated for urgent cases or for quality control as in blood banks, in which the set of tests is considered to be positive when at least one of the tests is positive, we found a sensitivity of over 99.96% for all pairs of tests (not including the blood culture test) and 100% when we consider ELISA, IIF, IHA tests together (Table 4).

The predictive values (positive and negative) of all combinationsof pairs of the five serological tests (ELISA, IIF, HAI, c-ELISA, rec-ELISA) confirm the presence of T. cruzi infection with a probability of100% in the stratum of donors with positive serology screening whenat least one of two tests has a positive result. In the stratum of donorswith negative serology screening results, it is possible to affirm theabsence of Chagas disease with a probability of 100% when at leastone of two tests shows negative results. However, in the stratum ofdonors with inconclusive serology it is possible to confirm the absenceof Chagas disease with a probability of 100% when the two tests havenegative results depending on the epidemiological variables (Table 5).

Discussion

In the present study, the general sensitivity rates were 97.76%, 97.70% and 97.64% and the specificity was 97.25%, 98.48% and 98.52% for the ELISA, IHA and IIF tests, respectively. These results show performances similar to those found by Langui Junior et al.⁽¹⁹⁾ but the sensitivity of IHA testing was higher in the present study. Studies on screening tests for Chagas disease reported sensitivity rates for the ELISA kit of bioMérieux ranging from 98.38% to 98.5% and specificity rates from 94.30% to 99.93%.^(20,21) Sensitivity rates ranging from 52.75% to 96.5% and specificity from 87% to 100%^(22-24) have been described for IIF. Sensitivity rates from 98.5% to 100% and specificity between 99.69% and 100% have been reported for IHA.^(19,20) Regarding the Bio-Manguinhos-FIOCRUZ kits, Gadelha et al.⁽²²⁾ found 100% sensitivity and specificity for the c-ELISA kit and sensitivity of 98.2% for the rec-ELISA kit.

Hence, it is essential to continually assess the performance of commercial tests that are still not totally efficacious. Failures in Chagas disease screening, may occur due to cross reactions with other parasites, in particular those of the trypanosomatid genus such as Leishmania which may have many genetic similarities with T. cruzi antigens and the same epitopes to bind to antibodiespresent in the sera of infected patients^(20,25).

The publication of government directives numbers 153 and 57 by ANVISA^(26,27), made a single technique for the serological screening of blood donors for Chagas disease mandatory and that the blood bank must participate in external quality control programs. Thus it is important to seek screening methods by investigating specific T. cruzi antigens which minimize or exclude cross reactions.

Cross reactions in blood banks may be a cause of persistentserological ineligibility due to Chagas disease; this may correspondto up to 80% of inconclusive or false-positive serological results.^{(8,11,20,28,29)} Many studies report that inconclusive samples repeatedlyshowed negative results on using other tests.^(11,26)

As, in this study, the estimated prevalence of Chagas disease was 13.30% in the stratum of donors with inconclusive serology at screening, 86.70% of donors probably do not have Chagas infection. This prevalence rate is well below the rate reported by Furuchó et al.⁽¹⁰⁾ who observed that 20.5% of donors in the inconclusive serology group were positive for Chagas disease. In addition, on considering epidemiological variables in this stratum, the absence of Chagas' infection can be affirmed with a probability of 100% when two tests give negative results in all pairs of serological tests.

Studies in epidemiology and statistics show that an associationof two tests increases the quality of diagnosis, thereby reducing thenumber of false results. The simplest way of forming a set of testsis by using in series or in parallel design⁽³⁰⁾ as used in this study.

Although there is no individual screening test for Chagas disease that has a sensitivity of 100%, sensitivity levels of 99.96% were found for several sets of tests in the parallel analysis. Additionally all pairs of tests (excluding HEMO) had specificities in series testing greater than 99.93% and the set formed by the ELISA, IIF and IHA tests had specificities of 100%, suggesting that this is the best alternative to confirm procedures.

Studies report that the combination of epidemiological data with results of high performance testing allows a more accurate view of the serologic status of donors^(10,11). To confirm these data, we find estimates for the sensitivity and specificity close to 100% for certain epidemiological covariates suggesting that it is important to consider the inclusion of covariates in the structure of the model to evaluate the performance of the diagnostic tests.

In conclusion, commercial diagnostic tests (ELISA, IIF, HAI, c-ELISA, rec-ELISA) used to screen for Chagas disease, when used in pairs in serial testing schemes, proved to be an interesting alternative to confirm the procedure. In addition, epidemiological variables may contribute to improve the results of these tests and to clarify the true meaning of inconclusive serology screening. In search of a gold standard procedure and more reliable estimates, further studies are necessary with larger samples, clinical variables and further assume dependence between tests in statistical modeling.

Acknowledgements

We thank the Fundação Hemominas for providing the data and the Brazilian funding agencies Capes, ANVISA and CNPq for financial support. We thank the Laboratory Bio-Manguinhos, especially Antônio Gomes Pinto, Yara de Miranda Gomes, Edimilson Domingos da Silva and Marcelle Bral de Mello for providing the c-ELISA and rec-ELISA kits as well as processing of samples from the respective techniques. We also thank the anonymous reviewers for their comments and criticisms.

Submitted: 9/16/2011

Accepted: 6/8/2012

Conflict-of-interest disclosure: The authors declare no competing financial interest

www.rbhh.org or www.scielo.br/rbhh

• ¹
  WHO 2010. First WHO report on neglected tropical diseases: Working to overcome the global impact of neglected tropical diseases (WHO/HTM/NTD/2010.1) 2010 [cited 2012 March 20]; Available from: http://whqlibdoc.who.int/publications/2010/9789241564090_eng.pdf
• 2. Organización Panamericana de la Salud. Estimación cuantitativa de la enfermedad de Chagas en las Americas. Geneva, Switzerland: OPAS; 2006. 28p.
• 3. Souza HM. Mecanismos alternativos de transmissäo parenteral da Doença de Chagas. Rev Méd Minas Gerais. 1993;3(2):93-5
• 4. Schmunis GA. Tripanosomíase Americana: seu impacto nas Américas e perspectivas de eliminação. In: Dias JC, Coura JR, organizadores. Clínica e terapêutica da doença de Chagas: uma abordagem prática para o clínico geral Rio de Janeiro: Fiocruz; 1997. p 11-24.
• 5. Dias JC. Elimination of Chagas disease transmission: perspectives Mem Inst Oswaldo Cruz. 2009;104(Suppl 1):41-5.
• 6. Schmunis GA. Epidemiology of Chagas disease in non-endemic countries: the role of international migration. Mem Inst Oswaldo Cruz. 2007;102(Suppl 1):75-85.
• 7. Salles NA, Sabino EC, Cliquet MG, Eluf-Neto J, Mayer A, Almeida-Neto C, et al. Risk of exposure to Chagas disease among seroreactive Brazilian blood donors. Transfusion. 1996;36(11-12):969-73.
• 8. Melo AS, Lorena VM, Moraes AB, Pinto MB, Leão SC, Soares AK, et al. The prevalence of chagasic infection among blood donors in the state of Pernambuco, Brazil. Rev Bras Hematol Hemoter. 2009;31(2):69-73.
• 9. Reesink HW. European strategies against the parasite transfusion risk. Transfus Clin Biol. 2005;12(1):1-4.
• 10. Furuchó, CR, Umezawa, ES, Almeida, I, Freitas, VL, Bezerra, R, Nunes EV, et al. Inconclusive results in conventional serological screening for Chagas' disease in blood banks: evaluation of cellular and humoral response. Trop Med Int Health. 2008;13(12):1527-33.
• 11. Ferreira-Silva MM, Pereira GA, Lages-Silva E, Moraes-Souza H. Socioepidemiological screening of serologically ineligible blood donors due to Chagas disease for the definition of inconclusive cases. Mem Inst Oswaldo Cruz. 2010;105(6):800-5.
• 12. Meira WS, Galvão LM, Gontijo ED, Machado-Coelho GL, Norris KA, Chiari E. Trypanosoma cruzi complement regulatory protein: a novel antigen for use in enzyme-linked immunosorbent assay for diagnosis of Chagas disease. J Clin Microbiol. 2002;40(10):3735-40.
• 13. Umezawa ES, Bastos SF, Coura JR, Levin MJ, Gonzalez A, Rangel-Aldao R, et al. An improved serodiagnostic test for Chagas disease employing a mixture of Trypanosoma cruzi recombinant antigens. Transfusion. 2003;43(1):91-7.
• 14. Umezawa ES, Luquetti AO, Levitus G, Ponce C, Henriquez D, Revollo S, et al. Serodiagnosis of chronic and acute Chagas disease with Trypanosoma cruzi recombinant proteins: results of a collaborative study in six Latin American countries. J Clin Microbiol. 2004;42(1):449-52.
• 15. Cheng KY, Chang CD, Salbilla VA, Kirchhoff LV, Leiby DA, Schohetman, Chat DO. Immunoblot assay using recombinant antigens as a supplemental test to confirm the presence of antibodies to Trypanosoma cruzi. Clin Vaccine Immunol. 2007;14(4):355-61.
• 16. Hadgu A, Qu Y. A Biomedical application of latent class models with random effects. Appl Stat. 1998;47(4):603-16.
• 17. Martinez EZ, Louzada-Neto F, Achcar JA, Syrjänen KJ, Derchain SF, Gontijo RC, et al. Bayesian Estimation of performance measures of screening tests in the presence of covariates: an absence of a gold standard. Braz J Probab Stat. 2009;23(1):68-81.
• 18. Hui SL, Walter SD. Estimating the error rates of diagnostic tests. Biometrics. 1980;36(1):167-71.
• 19. Langhi Junior DM, Bordin JO, Castelo A, Walter SD, Moraes-Souza H, Stumpf RJ. The application of latent class analysis for diagnostic test validation of chronic trypanosoma cruzi infection in blood donors. Braz J Infect Dis. 2002;6(40):181-7.
• 20. Flores-Chávez M, Cruz I, Rodríguez M, Nieto J, Franco E, Gárate T, et al. Comparación de técnicas serológicas convencionales y no convencionales para El diagnóstico de la enfermedad de Chagas importada en España. Enferm Infecc Microbiol Clin 2010;28(5):284-93. Spanish.
• 21. Praast G, Herzogenrath J, Bernhardt S, Christ H, Sickinger E. Evaluation of the Abbott ARCHITECT Chagas prototype assay. Diagn Microbiol Infect Dis. 2011;69(1):74-81.
• 22. Gadelha AA, Verçosa AF, Lorena VM, Nakazawa M, Carvalho AB, Souza WV, et al. Chagas disease diagnosis: comparative analysis of recombinant ELISA with conventional ELISA and the haemagglutination test. Vox Sang. 2003;85(3):165-70.
• 23. Pirard M, Iihoshi N, Boelaert M, Basanta P, López F, der Stuyft P. The validity of serologic tests for Trypanosoma cruzi and the effectiveness of transfusional screening strategies in a hyperendemic region. Transfusion. 2005;45(4):554-61.
• 24. Lunardelli A, Borges FP, Mello KF, Zeferino AS. Soroprevalência da doença de Chagas em candidatos a doadores de sangue. Rev Bras Anal Clin. 2007;39(2):139-41.
• 25. Brasil PE, Castro L, Hasslocher-Moreno Am, Sangenis L, Braga JU. Elisa versus PCR for diagnosis of chronic Chagas disease: Systematic review and meta-analysis. BMC Infect Dis. 2010;10:337-53.
• ²⁶
  ANVISA. Agência Nacional de Vigilância Sanitária. Resolução RDC nº 153, de 14 de junho de 2004. Publicada no Diário Oficial da união de 24 de junho de 2004. [citado 2001 Out 11]. Available from: http://e-legis.anvisa.gov.br/leisref/public/showAct.php?id=11662
• ²⁷
  ANVISA. Agência Nacional de Vigilância Sanitária. Resolução RDC nº 57, de 17 de outubro de 2010. Publicada no Diário Oficial da União em 16 de dezembro de 2010. [citado 2011 Out 11]. Available from: http://portal.anvisa.gov.br/
• 28. Leiby DA, Herron RM Jr, Garratty G, Herwaldt BL. Trypanosoma cruzi parasitemia in: US Blood donors with serologic evidence of infection. J Infect Dis. 2008;198(4):609-13
• 29. Sabino EC, Salles NA, Sarr M, Barreto AM, Oikawa M, Oliveira CD, Leao SC, Carneiro-Proietti AB, Custer B, Busch MP; NHLBI Retrovirus Epidemiology Donor Study-II (REDS-II), International Component. Enhanced classification of Chagas serologic results and epidemiologic characteristics of seropositive donors at three large blood centers in Brazil. Transfusion. 2010;50(12):2628-37.
• 30. Arango HG. Bioestatística: teórica e computacional. 2nd ed. Rio de Janeiro: Guanabara Koogan; 2005.

Publication Dates

• Publication in this collection
  06 Sept 2012
• Date of issue
  2012

History

• Received
  16 Sept 2011
• Accepted
  08 June 2012